Abstract: Meningitis-like epidemic diseases is the largest threat to the frog farming industry. The causing pathogen, Elizabethkingia miricola, often exhibits extensive antibiotic resistance, but the resistance mechanisms remain poorly understood. Our aim was to examine the antimicrobial susceptibility and biofilm forming ability of the E. miricola strains isolated from frogs, and to determine their mechanisms of β-lactams and fluoroquinolones resistance. Isolates were re-identified to precise species using 16S rRNA and rpoB gene phylogenetic tree. Antimicrobial susceptibility, biofilm formation, and the minimum biofilm eradication concentration (MBEC) were examined by using broth dilution method, crystal microtiter plate assay, and the calgary biofilm device (CBD), respectively. β-lactamase genes and mutations in the quinolone-resistance determining regions (QRDR) were identified using PCR. ESBLs and MBLs production were screened using a combined disc test. All dominant strains in diseased frogs collected from different farms were identified as E. miricola. The isolates were resistant to β-lactams, aminoglycosides, and fluoroquinolones tested, whereas all isolates were susceptible only to doxycycline and florfenicol among 17 antibiotics. The positive rate of biofilm formation was 100% among the 9 E. miricola isolates, with the majority being low to moderate biofilm formers. The MBEC of antibiotics tested on biofilm bacteria were 8—256 times higher than the MIC on planktonic bacteria. All strains carried β-lactam resistance genes (blaCME, blaGOB, and blaB), ESBL and MBL phenotypes were observed for all isolates. A single mutation was found in the gyrA gene (S83R), which confer fluoroquinolone resistance, while no mutations were found in the gyrB, parC, or parE gene of these isolates. The results indicate that frog-derived E. miricola exhibits multidrug resistance and generally has the ability to form biofilms. The production of β-lactamase and mutation at QRDR correspond with its β-lactam and fluoroquinolone resistance phenotypes. Doxycycline and florfenicol exhibited good sensitivity in vitro and have the potential for treating severe E. miricola infections.