The present study aimed to investigate whether different citrus peel powders (orange, mandarin, and lemon) could be incorporated into polylactic acid (PLA), for the preparation and characterization of biodegradable films made of this fruit waste. In particular, films were prepared by the solvent casting method and tested in terms of physicochemical and biochemical parameters, along with mechanical properties. Furthermore, the citrus peel powders were tested for their physicochemical and biochemical properties after ethanolic extraction. The results showed that the addition of citrus powders into PLA proved to be efficient compared to the control films (PLA). The citrus peel-based films showed high antioxidant capacity (38.37%–32.54%), a considerable total phenolic content (17.2–12.58 mg GAE/L), good lightness (L*: 42.83–41.93), elastic Young’s modulus (257.95–175.38 MPa), oxygen transmission rate (976.5 mL m⁻² day⁻¹–1218 mL m⁻² day⁻¹), elongation at break (183.06%–135.95%), and tensile strength (6.15–4.56 MPa), which were affected significantly (p < 0.05) by the botanical origin of citrus peel. Concerning the citrus peel extracts, the highest values in antioxidant capacity (52.56%), total phenolic content (724.8 mg GAE/L), and titratable acidity (714.67 mg of citric acid/L) were recorded in the extract of lemon peel powders. The corresponding values for orange peel powder extracts were 39.43%, 563.8 mg GAE/L, and 576 mg of citric acid/L, while those for mandarin were 33.01%, 558.46 mg GAE/L, and 54.67 mg of citric acid/L. The application of multivariate analysis of variance and linear discriminant analysis on the mechanical properties, physicochemical, and biochemical data resulted in the differentiation of pure PLA and PLA-based films fabricated with citrus peel powder. From the above findings, it is concluded that citrus peel powders are sources of phytochemicals and can be used for the preparation of PLA-based films for potential applications in food preservation.