Tilapia (Oreochromis niloticus), which is extensively farmed globally and ranks as the second most cultivated fish in the Philippines, generates significant amounts of waste that are often underutilized. One specific type of waste material consists of fish heads, which contain a valuable source of extracellular matrix (ECM). This study aims to evaluate the effects of sonication as a viable decellularization method for the extraction of ECM from tilapia fish heads. Particularly, two treatments were tested on the head samples: sonication-assisted decellularization (dWS) using a water bath sonicator, and decellularization without sonication (dNS), each with different contact times (5 min and 10 min). Histological analysis with H and E staining and DNA quantification revealed that sonication-assisted samples (dWS) showed a greater reduction in basophilic components and DNA content, achieving a 93.7% removal rate. These dWS samples also had the highest protein loss, retaining only 33.86% of the original protein. SDS–PAGE analysis indicated that both dWS and dNS samples maintained similar collagen structures, as evidenced by identical subunit bands. ATR–FTIR spectra confirmed the presence of collagen type I in all samples, detecting characteristic amides A, B, I, II, and III. The results revealed that varying treatments and contact times had significant effects on the physical and mechanical properties of the decellularized extracellular matrix (ECM). These findings highlight the effectiveness of sonication in the decellularization process, particularly for utilizing waste tilapia heads.