Ficolins have crucial functions in recognizing and eliminating pathogens in innate immunity. In this study, we identified two ficolin-like genes from the oriental river prawn, Macrobrachium nipponense. These genes were designated as MnFico1 and MnFico2. MnFico1 cDNA has 1600 bp, whereas MnFico2 has 1486 bp. In addition to a coiled-coil region or a low complexity region, the two ficolins both contained a signal peptide and a fibrinogen-related domain. qRT-PCR results showed that the highest expression level of MnFico1 expression was in the gills, whereas that of MnFico2 was in the heart. The transcripts of MnFico1 and MnFico2 could both respond to bacteria challenge. The transcription of antilipopolysaccharide factors ALFs (MnALF1 and MnALF2) and crustin genes (MnCru4, MnCru5, MnCru6, and MnCru7) was inhibited in the gills of MnFico1 or MnFico2 knockdown prawns at 24 h Vibrio parahaemolyticus challenge. Recombinant proteins of rMnFico1 and rMnFico2 could bind toward diverse bacteria and agglutinate Gram-negative and Gram-positive bacteria with the presence of calcium (Ca2+). rMnFico1 and rMnFico2 proteins also have lipopolysaccharide and peptidoglycan binding activity. Both recombinant ficolin proteins could help the prawn to facilitate the clearance of V. parahaemolyticus in vivo. Our results suggested that MnFico1 and MnFico2 might serve as pattern recognition receptors in M. nipponense.