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This study aims to produce, characterize, and assess the antimicrobial activity and cytotoxicity of polymer blends based on chitosan (CT) and fish collagen (COL) produced by different precipitation methods. Polymer blends were obtained in alkaline (NaOH), saline (NaCl), and alkaline/saline (NaOH/NaCl) solutions with different CT:COL concentration ratios (20:80, 50:50, and 80:20). The polymer blends were characterized by various physicochemical methods and subsequently evaluated in terms of their in vitro antimicrobial and cytotoxicity activity. In this study, the degree of chitosan deacetylation was 82%. The total hydroxyproline and collagen content in the fish matrix was 47.56 mg. g−1 and 394.75 mg. g−1, respectively. The highest yield was 44% and was obtained for a CT:COL (80:20) blend prepared by precipitation in NaOH. High concentrations of hydroxyproline and collagen in the blends were observed when NaOH precipitation was used. Microbiological analysis revealed that the strains used in this work were sensitive to the biomaterial; this sensitivity was dose-dependent and increased with increasing chitosan concentration in the products. The biocompatibility test showed that the blends did not reduce the viability of fibroblast cells after 48 h of culture. An analysis of the microbiological activity of the all-polymer blends showed a decrease in the values of minimal inhibitory concentration (MIC) and minimal bactericidal concentrations (MBC) for S. aureus and P. aeruginosa. The blends showed biocompatibility with NIH-3T3 murine fibroblast cells and demonstrated their potential for use in biomedical applications such as wound healing, implants, and scaffolds. Key points: Different precipitation methods do not change the biological properties of polymer blends. Gram-positive bacterias and Pseudomonas aeruginosa were sensitive to polymer blends. The blends produced showed biocompatibility in NIH-3T3 murine fibroblast cells. [ABSTRACT FROM AUTHOR]