The resistance of bacteria to antibiotics is a major public health issue. Klebsiella pneumoniae is a type exemplification of multi-resistant enterobacteria. Its high biofilm forming capacity is a major factor in the recurrent infection of the intestinal tract. In this study, the intrinsic mechanism of secondary growth of K. pneumoniae in response to antibiotics and the inhibition effect of probiotic supernatant on biofilm formation after antibiotic treatment were investigated in a polyester nonwoven chemostat bioreactor. The experimental results showed that the c-di-GMP content in the cells increased after treatment with levofloxacin, leading to the formation of a thick biofilm due to an increase in the production of extracellular polymer substance (EPS) and type 3 fimbriae. Biofilm prevents the mass transfer of levofloxacin and protects K. pneumoniae cells from being killed by levofloxacin. Under suitable conditions, K. pneumoniae cells on the biofilm enter into the suspension for secondary growth. Moreover, the inhibition of probiotic supernatant on the biofilm formation was mainly due to the reduced expression of yfiN and mrkJ genes, and the decreased concentration of c-di-GMP in cells, as well as the less secretion of EPS. At the same time, the decrease in the concentration of c-di-GMP also reduced the expression of the mrkABCDF gene and prevented the synthesis of the type 3 fimbriae. The results would help to understand the mechanism of antibiotic resistance of pathogenic bacteria and to provide evidence to address this problem through the use of probiotics.