<p><i>Vibrio cholerae</i> is a human pathogen transmitted through the consumption of contaminated food or water. The O1 and O139 serogroups can lead to cholera outbreaks and epidemics, while non-O1/non-O139 serogroups can cause sporadic moderate or mild diarrhea and can also cause systemic infections. In this study, we performed genomic sequencing and phenotypic assays on 13 non-O1/non-O139 <i>V. cholerae</i> isolates (7 clinical and 6 environmental strains). Our results revealed that, except for the environmental isolate NOVC12, most clinical and environmental strains clustered separately on the phylogenetic tree. All isolates lacked the virulence genes <i>ctxAB</i> and <i>tcpA</i>, but harbored potential virulence factors including <i>hlyA</i>, <i>gbpA</i>, and <i>rtxA</i>. All strains may encode the type VI secretion system, while most clinical isolates and two environmental isolates additionally harbor the component genes of the type III secretion system. Five clinical isolates and two environmental isolates likely harbor integrative and conjugative elements. Phenotypic assays confirmed that these strains exhibited hemolytic activity, biofilm formation, and motility, and all strains were susceptible to tetracycline and azithromycin. The results of this study provide insights into the characteristics of clinical and environmental isolates of non-O1/non-O139 <i>V. cholerae</i>. Surveillance of these strains is of great significance for the prevention and control of disease infections.</p>