AbstractProteinase K (ProK) is regarded as an essential ingredient in most DNA extraction protocols for protein‐rich sample types such as tissue, blood, and mucus. However, ProK is expensive and may be unnecessary when samples are protein‐limited, such as environmental DNA (eDNA) from oligotrophic seawater. To investigate this, we filtered seawater through Sterivex cartridges from a mesocosm receiving input from the adjacent coral reef slope at the Hawai‘i Institute of Marine Biology. We tested whether the addition of varying levels of ProK (0 μL, 25 μL, 50 μL, 100 μL, or 150 μL—20 mg/mL stock concentrations) to 1.8 mL of lysis buffer affected DNA yield and the diversity, community composition, and detection of known organisms within the mesocosm community based on zero‐width operational taxonomic units (ZOTUs) obtained from DNA metabarcoding. We found no significant differences in diversity metrics among ProK quantities and dominant ZOTUs were consistent across concentrations. Over 50% of detected ZOTUs were shared among samples and only two ZOTUs were unique to samples of a particular ProK concentration. While the community composition among ProK quantities differed, pairwise community comparisons between quantities were not statistically significant and matched the known species composition of the mesocosm. These results suggest that rare ZOTUs and eDNA patchiness are driving overall community differences as opposed to extraction ingredients. Our data show that ProK is not essential when assessing communities from oligotrophic marine environments using eDNA and that the reduction or elimination of ProK can decrease sample preparation time and costs while maintaining data integrity.