Black rockfish (Sebastes schlegelii), an important aquaculture species in Korea, has been affected by bacterial diseases leading to a drastic decline in production. Goose-type lysozyme (LysG) is a key enzyme of the innate immune system to eradicate bacterial infections. In this study, two isoforms of LysG from black rockfish, designated as RfLysG1 and RfLysG2, have been identified and characterized at the molecular, transcriptional, and functional levels. The deduced amino acid sequences had the LysG family characteristics and exhibited conserved properties, including active residues and domains. The cDNA sequences of RfLysG1 and RfLysG2 were 1514 bp and 900 bp in length, respectively. The 567-bp open reading frame (ORF) of RfLysG1 encoded a protein of 188 amino acids with molecular mass 20.11 kDa, and the 600-bp ORF of RfLysG2 encoded a polypeptide with 199 amino acids and molecular mass of 22.19 kDa. Homology studies indicated that RfLysG1 showed the highest identity (84.6%) with LysG-B of Oplegnathus fasciatus, while RfLysG2 showed the highest identity (74.4%) with LysG of Siniperca chuatsi. Both sequences possessed a soluble lytic trans-glycosylase domain. Both lacked signal peptide and they were not identified as proteins secreted by non-classical pathway by the SecretomeP server. Transcriptional analysis of the two genes showed constitutive expression, where both genes were highly expressed in blood under normal physiological conditions. In response to the immune challenges lipopolysaccharide (LPS), Streptococcus iniae, and poly I:C injection, the expression of RfLysG1 and RfLysG2 was significantly upregulated in blood and spleen tissues in a time-dependent manner. Turbidimetric assays indicated that both recombinant proteins tagged with maltose-binding protein (MBP) were reactive against several Gram-positive and Gram-negative bacteria, but MBP was inactive. Optimum temperatures for the recombinant RfLysG1 and RfLysG2 were 40 °C and 50 °C, respectively, and both were highly active at pH 3.0. The results provide evidence for the vital immunological role and bacteriolytic potential of RfLysG1 and RfLysG2.