The search for bioactive peptides with therapeutic properties has promoted the use of marine byproducts as collagen sources. This study evaluated the effect of collagen hy-drolysates (CH) obtained from the swim bladder (SB) of Totoaba macdonaldi on breast cancer cells (MCF-7) and human dermal fibroblasts (CRL-1474), considering the need for less invasive and less toxic treatment alternatives. Two pretreatment methods for the SB were compared: (1) NaOH and butanol (SBPT), and (2) hexane (SBDF). The pretreated tissues underwent direct enzymatic hydrolysis using bromelain. The resulting hydrolysates were characterized by SDS-PAGE, Raman spectroscopy, and chromatographic profiling. Both pretreatments preserved the structure of type I collagen. Bromelain hydrolysis was efficient, yielding peptides with molecular weights below 20 kDa for CH-SBPT and below 10 kDa for CH-SBDF. CH-SBDF showed greater hydrophilicity and significantly reduced MCF-7 cell viability, particularly at 20 mg/mL, with a clear dose-response relationship. In CRL-1474 fibroblasts, CH-SBDF stimulated cell proliferation, while CH-SBPT had neutral effects. Hexane pretreatment is a viable alternative to NaOH, reducing processing steps without compromising yield or bioactivity. CH derived from Totoaba macdonaldi exhibit promising anticancer and regenerative properties, suggesting potential biomedical ap-plications. Further research is needed to isolate active peptides and elucidate their mechanisms of action.