Polyhydroxyalkanoates (PHAs) are microbial-derived biodegradable polymers with significant potential to replace conventional synthetic plastics. This study investigates the novel biosynthesis of PHAs by Paramecium caudatum, a ciliated protozoan, using lignocellulosic waste sugarcane bagasse (SCB) as a low-cost carbon source. Paramecium sp. was isolated from industrial wastewater and identified as P. caudatum via 18S rRNA gene sequencing (GenBank: PQ038083). P. caudatum cultures were incubated separately with SCB at 4% (w/v) and glucose at 3% (w/v) for comparison, under optimal conditions (25°C, pH 7). PHA accumulation was assessed using Sudan Black B and Nile Blue A staining during the log phase. Extracted PHAs were characterized using Fourier-transform infrared spectroscopy (FTIR), gas chromatography–mass spectrometry (GC–MS), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). PHA synthase expression in total cellular protein was assessed via SDS-PAGE, and PHA film biodegradability was evaluated through a soil burial test. PHA yields reached 2.43 g/L with glucose and 2.21 g/L with SCB. FTIR and GC–MS analyses revealed predominant PHA monomers, including 3-hydroxybutyrate ethyl ester (92.6%), hexadecanoic acid ethyl ester (74.4%), and octadecanoic acid ethyl ester (65.9%). TGA showed good thermal stability, with Tmax at 280°C (glucose) and 270°C (SCB). SEM imaging displayed a porous, pseudospherical surface morphology, indicating strong polymer integrity. The PHA films exhibited favorable plasticizing properties and fully biodegraded within 30 days in soil. SDS-PAGE confirmed consistent expression of a ~63 kDa PHA synthase under both carbon source conditions. This is the first study to report P. caudatum as a microbial cell factory for scalable bioplastic production via lignocellulosic waste valorization, contributing to circular bioeconomy goals.