To ascertain upon the ideal configuration of physico-mechanical qualities, efficient processing techniques, and network stability of the prepared bio-composite films in real-world applications, the polymeric materials shall be subjected to a careful manipulation. Such bio-composite films have outstanding combinations of biocompatibility and toxicity-associated safety qualities. Such research interventions will be beneficial for the packaging, pharmaceutical, and biomedical industries that wish to target and adopt them for commercial applications. In this article, three alternate organic acids, i.e., citric acid (CA), tartaric acid (TA), and malic acid (MA), are blended separately into polyvinyl alcohol (PVA)-starch (St)-glycerol (Gl) composite films and for the targeted purpose of enhanced crosslinking, plasticizing, and antibacterial capability of the polymer network. The organic acid-based bio-composite polymeric films were assessed in terms of swelling index (SI), in vitro degradation, tensile strength (TS), percentage elongation (%E), antibacterial activity, and cytotoxicity attributes. Among these, the MA-based PVA composite films outperformed the CA-based PVA composite film in terms of absorbency (SI 739.29%), mechanical strength (TS 4.88 MPa), and elasticity (%E 103.68%). Furthermore, following a 24-h incubation period, the MA-based films exhibited the highest proliferative effect of 215.59% for the HEK cells. In conclusion, the MA has been inferred to be the most relevant organic acid for the desired optimality of film composition, physical and biological properties, and cost.