One of the main reasons for the bacterial resistance to antibiotics is caused by biofilm formation of microbial pathogens during bacterial infections. Salmonella enterica and Vibrio harveyi are known to form biofilms and represent a major health concern worldwide, causing human infections responsible for morbidity and mortality. The current study aims to investigate the effect of purified sulfated polysaccharides (SPs) from Chlamydomonas reinhardtii (Cr) on planktonic and biofilm growth of these bacteria. The effect of Cr-SPs on bacterial planktonic growth was assessed by using the agar well diffusion method, which showed clear zones ranging from 13 to 26 mm in diameter from 0.5 to 8 mg/mL of Cr-SPs against both the bacteria. Time-kill activity and reduction in clonogenic propagation further help to understand the anti-microbial potential of Cr-SPs. The minimum inhibitory concentration of Cr-SPs against S. enterica and V. harveyi was as low as 440 mu g/mL and 490 mu g/mL respectively. Cr-SPs inhibited bacterial cell attachment up to 34.65-100% at 0.5-8 mg/mL in S. enterica and V. harveyi respectively. Cr-SPs also showed 2-fold decrease in the cell surface hydrophobicity, indicating their potential to prevent bacterial adherence. Interestingly, Cr-SPs efficiently eradicated the preformed biofilms. Increased reduction in total extracellular polysaccharide (EPS) and extracellular DNA (eDNA) content in a dose-dependent manner demonstrates Cr-SPs ability to interact and destroy the bacterial EPS layer. SEM analysis showed that Cr-SPs effectively distorted preformed biofilms and also induced morphological changes. Furthermore, Cr-SPs also showed anti-quorum-sensing potential by reducing bacterial urease and protease activities. These results indicate the potential of Cr-SPs as an anti-biofilm agent and will help to develop them as alternative therapeutics against biofilm-forming bacterial infections.