As novel amphiphilic materials, six uncharged alkyl rhamnosides incorporating various alkyl chain and one rhamnose amine quaternary ammonium salt were successfully synthesized in this study. Their amphiphilic properties (HLB and CMC), antimicrobial and anti-biofilm activity against S. aureus and P. aeruginosa were investigated. Differentially regulated proteins and pathways were identified by comparative proteomics research to first give a sight on how alkyl rhamnosides performed the anti-biofilm activity at protein and pathway levels. Among the uncharged alkyl rhamnosides, dodecyl rhamnoside and octyl rhamnoside showed the best antimicrobial and anti-biofilm ability against S. aureus and against P. aeruginosa, respectively. Interestingly, the relationships between amphiphilic properties or MIC with anti-biofilm activity were first established. Uncharged alkyl rhamnoside with an optimized HLB value of 5.0 had both the strongest antibacterial and anti-biofilm activity against S. aureus, and MIC was the maximum biofilm inhibitory concentration for all alkyl rhamnosides. Alkyl rhamnosides have a significant overall regulatory effect on the proteomics and pathways of bacterial biofilms, including energy production, substrates transportation, signal transduction, key molecules balance, and so on. These amphiphilic materials have a great potential to be used as additives in pharmaceutic, cosmetic, food industry, hospital and in other non-medical fields.