Wastewater treatment plants (WWTPs) harbor bacteria and antimicrobial resistance genes, favoring gene exchange events and resistance dissemination. Here, a culture-based and metagenomic survey of qnrA, qnrB, qnrS, and aac(6′)-Ib genes from raw sewage (RS) and activated sludge (AS) of a full-scale municipal WWTP was performed. A total of 96 bacterial isolates were recovered from nalidixic acid-enrichment cultures. Bacteria harboring the aac(6′)-Ib gene predominated in RS, whereas qnrS-positive isolates were specific to AS. Novel qnrS- and aac(6′)-Ib-cr positive species were identified: Morganella morganii, Providencia rettgeri, and Pseudomonas guangdongensis (qnrS), and Alcaligenes faecalis and P. rettgeri (aac(6′)-Ib-cr). Analysis of qnrS and aac(6′)-Ib sequences from isolates and clone libraries suggested that the diversity of qnrS is wider than that of aac(6′)-Ib. A large number of amino acid mutations were observed in the QnrS and AAC(6′)-Ib proteins at previously undetected positions, whose structural implications are not clear. An accumulation of mutations at the C72, Q73, L74, A75 and M76 positions of QnrS, and D181 of AAC(6′)-Ib might be important for resistance. These findings add significant information on bacteria harboring qnrS and aac(6′)-Ib genes, and the presence of novel mutations that may eventually emerge in clinical isolates.