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A significant challenge in the design of biomimetic scaffolds is combining morphologic, mechanical, and biochemical cues into a single construct to promote tissue regeneration. In this study, we analyzed the effects of different crosslinking conditions on fibrin biopolymer microthreads to create morphologic scaffolds with tunable mechanical properties that are designed for directional cell guidance. Fibrin microthreads were crosslinked using carbodiimides in either acidic or neutral buffer, and the mechanical, structural, and biochemical responses of the microthreads were investigated. Crosslinking in the presence of acidic buffer (EDCa) created microthreads that had significantly higher tensile strengths and moduli than all other microthreads, and failed at lower strains than all other microthreads. Microthreads crosslinked in neutral buffer (EDCn) were also significantly stronger and stiffer than uncrosslinked threads and were comparable to contracting muscle in stiffness. Swelling ratios of crosslinked microthreads were significantly different from each other and uncrosslinked controls, suggesting a difference in the internal organization and compaction of the microthreads. Using an in vitro degradation assay, we observed that EDCn microthreads degraded within 24h, six times slower than uncrosslinked control threads, but EDCa microthreads did not show any significant indication of degradation within the 7-day assay period. Microthreads with higher stiffnesses supported significantly increased attachment of C2C12 cells, as well as increases in cell proliferation without a decrease in cell viability. Taken together, these data demonstrate the ability to create microthreads with tunable mechanical and structural properties that differentially direct cellular functions. Ultimately, we anticipate that we can strategically exploit these properties to promote site-specific tissue regeneration.