Enzymatic hydrolysis is one of the effective methods used to obtain the bioactive peptides from marine resources. This study aimed to evaluate effect of the enzyme type (Food Pro PNL (FP), Corolase8000 (C8), and Corolase7089 (C7)) and biomass pretreatment level (whole starfish (SF), deproteinized (DPSF) as well as deproteinized and demineralized starfish (DPDMSF)) on the hydrolysate yield, degree of hydrolysis (DH), generated peptides’ molecular weight (MW), and in vitro radical scavenging and antiproliferative effects. Regardless of the enzyme used, deproteinization reduced the hydrolysate yield (<8% dw/ww) and DH (40%) to SF. However, the protein content of hydrolysates from DPSF and DPDMSF was higher than that prepared from SF. C8 was not effective in hydrolyzing SF but was the only effective enzyme in hydrolyzing DPDMSF. The peptides’ MW distribution strongly depended on the pretreatment and enzyme type, mostly ranging from 17 to 70 kDa. Glycine content was higher in hydrolysates from DPSF and DMDPSF, indicating their collagenous nature. Hydrolysates from DPSF, rich in collagenous peptides, showed medium MW but the highest radical scavenging activity. Only SF-FP hydrolysate, rich in non-collagenous peptides, showed antiproliferative activity against melanoma cancer cells. Overall, the findings demonstrate that upstream biomass pretreatment and enzyme selection directly govern the yield and bioactivity of starfish protein hydrolysates, providing a rational basis for designing starfish protein hydrolysates with targeted functional properties.