Abstract: This study aims to explore the preparation and properties of seedling trays with large-size straw fibers. The wheat straw was selected as the raw material. Four pretreatments were applied to obtain the large-size straw fibers, including the biological pretreatment, biological pretreatment plus MnO2, biological pretreatment plus KOH, and biological pretreatment plus MnO2 and KOH. The corresponding fibers were then labeled as the CS, CSM, CSK, and CSMK. Seedling trays were produced through negative pressure suction filtration. The straw fibers were characterized after different pretreatments. A systematic investigation was implemented to explore their effects on the preparation and properties of seedling trays. The results showed that the average lengths of CS, CSM, CSK, and CSMK fibers were 2.20, 1.95, 1.70, and 1.55 mm, respectively, and the average widths were 96, 94, 94, and 86 µm, respectively. Mechanical properties of seedling trays showed that the dry and wet tensile strengths increased with the fibers that were pretreated with MnO2 or/and KOH. CSMKT trays exhibited the highest mechanical strength among all tested samples. The dry tensile strength of CSMKT fiber was 128% greater than that of CS fibers, and the wet tensile strength was approximately twice. In terms of water resistance, the maximum water absorption rates of the various trays after 240 min were 629% for CST, 519% for CSMT, 524% for CSKT, and 523% for CSMKT, respectively. The results indicated that the seedling trays fabricated from the straw fibers treated with MnO2 and/or KOH also exhibited superior bonding properties, resulting in enhanced structural integrity under water conditions. The test results showed that significant dissolution and swelling were observed for the CST trays. Furthermore, the relatively looser structure was observed in the CST trays, whereas, the more tightly bonded one was found in the CSMT, CSKT, and CSMKT samples. Notably, the lowest dissolution and swelling rates were observed for the CSMKT, indicating its enhanced structural cohesion and dimensional stability. In particular, the thickness swelling rate of CSMKT was 1.26%, which was more than 50% lower than that of CST trays. As such, the CSMKT can be expected to maintain structural integrity under varying moistures. A seedling nursery test was conducted using CSMKT as the experimental group and the plastic seedling trays as the control. It was found that the seedlings grew better in the straw fiber-based trays, where the seedling strength index of cucumber, tomato, croissant, and chili pepper increased by 46.67%, 61.54%, 76.92%, and 171.43%, respectively, compared with the control group. Besides, the SPAD value and root vitality of the seedlings in the experimental group were generally higher by more than 10% and 38%, respectively, compared with the plastic trays. There was a low production cost of the seedling trays with the large-size straw fiber under different pilot conditions, regarding the material and energy consumption. The production cost also ranged from 0.49 to 0.51 yuan/piece, indicating better economic viability. The large-size wheat straw fibers after biological fermentation coupled with mechanical disc milling can be expected to serve as the seedling tray by suction filtration. The addition of MnO2 or/and KOH on the basis of biological fermentation can significantly promote the deaggregation of fiber bundles, leading to the more uniform distribution of fibers. The finding is more conducive to the mechanical properties and water resistance of the seedling trays.