||Fishery by-products (FBPs) have been increasingly investigated for the extraction and production of a vast array of active molecules. The aim of this study was to produce phenazine compounds from FBPs via microbial fermentation and assess their novel antinematode effect. Among various FBPs, squid pen powder (SPP) was discovered as the most suitable substrate for phenazine production by Pseudomonas aeruginosa TUN03 fermentation. Various small-scale experiments conducted in flasks for phenazine production indicated that the most suitable was the newly designed liquid medium which included 1% SPP, 0.05% MgSO4, and 0.1% Ca3(PO4)2 (initial pH 7). Phenazines were further studied for scale-up bioproduction in a 14 L bioreactor system resulting in a high yield (22.73 µg/mL) in a much shorter cultivation time (12 h). In the fermented culture broth, hemi-pyocyanin (HPC) was detected as a major phenazine compound with an area percentage of 11.28% in the crude sample. In the bioactivity tests, crude phenazines and HPC demonstrate novel potential nematicidal activity against black pepper nematodes, inhibiting both juveniles (J2) nematodes and egg hatching. The results of this work suggest a novel use of SPP for cost-effective bioproduction of HPC, a novel potential nematodes inhibitor. Moreover, the combination of MgSO4 and Ca3(PO4)2 was also found to be a novel salt composition that significantly enhanced phenazine yield by P. aeruginosa fermentation in this work.