Purified fungal chitosan and crustacean chitosan were wet spun by using adipic and lactic acids as solvent. The lowest viscosity at which fiber formation was possible was 0.5 Pa·s; below this value, aggregates from low molecular weight fungal chitosan (32 kDa) formed, which could not be collected and dried. Fiber formation was achieved with high molecular weight fungal (400 kDa) and shrimp (406.7 kDa) chitosans as well as low molecular weight shrimp chitosan (50–190 kDa). Fibers made of high molecular weight chitosans with adipic acid as the solvent generally exhibited higher tensile strength; the highest observed tensile strength and Young’s modulus were 308.0 ± 18.4 MPa and 22.7 ± 4.0 GPa, respectively. SEM images indicated the formation of cylindrical chitosan fibers. The survival (viability) of human skin fibroblasts in presence of different fibers was measured using tetrazolium-based colorimetric assay and results confirmed that chitosan fibers have better biocompatibility than common conventional sutures, regardless of the chitosan and acid type. Accordingly, chitosan fibers from fungal and shrimp sources serve as good candidates for application as sutures.