Scales of Prochilodus magdalenae, a Colombian endemic fish species, were used to obtain chitosan for application as an antibacterial agent integrated into starch-based films. Analysis of its composition during the demineralization and deproteinization process indicated that minerals and protein were both removed successfully. At this point, mild conditions for the deacetylation process were employed, namely, 2, 4, and 6 wt.% NaOH at room temperature for 16 h. Chitosan processed under 2 wt.% NaOH had low molecular weight, with the lowest value of 107.18 ± 24.99 kDa, which was closely related to its antibacterial activity. Finally, this chitosan was integrated into a banana starch-based film, and its antibacterial activity was assayed in Escherichia coli and Staphylococcus aureus cultures, with positive results in the former culture, especially due to the low-molecular-weight characteristic of chitosan.