In this work, films based on bacterial cellulose nanofibers (BCNFs) incorporating gelatin hydrolysate (GH) from tilapia skin were produced. The effect of plasticizer (sorbitol or glycerol) and GH incorporation was evaluated on the physical-chemical and optical properties of films. BCNFs were produced using bacterial cellulose obtained from Hestrin and Schramm (HS) medium (BCNF-HS) or cashew apple juice (BCNF-CM), which was studied as an alternative to HS. Films with sorbitol showed the best properties and were selected for further characterization, using 40% (w/w) of BCNF-HS, 40% (w/w) of GH and 20% (w/w) of sorbitol (BCNF-HS-S-GH films). These films exhibited an antioxidant activity of 7.8 lmols Trolox Eq/g film, a water vapor permeability (WVP) of 1.6 g.mm/kPa.h.m 2 and an Young's modulus of 0.57 GPa. Films produced with BCNFs obtained from cashew apple juice revealed enhanced tensile strength, elongation at break, and thermal stability. Caco-2 cells' viability after incubation with BCNF-based films incorporating GH was evaluated and showed non-cytotoxicity, reinforcing the safety of the developed materials and their potential use in food applications.