In this study, Botryocladia leptopoda was analyzed to identify proteins and bioactive peptides. First, the proteins expressed by Botryocladia leptopoda were extracted using enzymatic and alkali treatments and identified using proteomics. Two proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and phycoerythrin beta chain, were chosen for the analysis of potential bioactive peptides based on phylogenetic relationships and the higher sequence coverage. The two identified protein sequences were subjected to potential active peptide analysis and simulated enzyme hydrolysis using the BIOPEP-UWM database. The results showed Botryocladia leptopoda was predicted to generate peptides with angiotensin-converting enzyme (ACE) inhibitory and antioxidant activities. The simulated enzymatic hydrolysis of Botryocladia leptopoda using 6 different enzymes showed that the pepsin hydrolysate produced peptides with the highest ACE inhibitory activities (97.65% ± 1.78%) compared with the other hydrolysates, which was consistent with the BIOPEP-UWM simulation results. The predicted ACE inhibitory activity of the pepsin hydrolysate is not significantly different from that of a commercial hypertension drug (captopril) used clinically, and the pepsin hydrolysate activity was not affected by pH. In addition, the peptides derived from the pepsin hydrolysate with molecular weights ≥10 kDa showed the best ACE inhibitory activity and nitric oxide contents compared with peptides with molecular weights of 3–10 kDa and ≤3 kDa, respectively. This study demonstrates that a proteomics approach combined with the BIOPEP-UWM database can be a powerful tool to assist with the preparation of functional hydrolysates or peptides. This study also revealed that protein hydrolysates generated from Botryocladia leptopoda contain peptides with ACE inhibitory activities, especially those in the molecular weight ≥10 kDa fraction, which can be used as a natural material for blood pressure management. The ACE inhibitor peptide from Botryocladia leptopoda proteins could be purified and the sequence determined in future studies to develop functional supplemental foods.