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The efficiency of osmotic backwashing cleaning to remove bacteria from forward osmosis membranes was systematically studied for the first time under different attachment and osmotic backwashing conditions. It is hypothesized that biofouling is preventable when tackling initial adhesion, i.e. during the reversible stage. Cell removal from the membrane was dependent on both adhesion and backwashing conditions: tests were performed for backwashing solutions of different concentrations and salt type, as well as different filtration durations and Ca2+ concentrations in the feed solution. Following adhesion of P. putida, a backwashing draw solution (DSobw) of 3 M NaCl was the most efficient, removing 93% of the adhered cells after 1 min of backwashing. All adhered cells left on the membrane were dead/injured due to osmotic shock. To optimise the cleaning regime, the maximum filtration time for which backwashing is efficient must be determined. This was determined to be 30 min, after which backwashing became inefficient, only removing 78% of cells. The addition of 5 mM Ca2+ to the feed caused a 50% increase in cell surface coverage compared to adhesion without Ca2+. This increase in adhesion rendered backwashing inefficient, as cell removal was only 60%. To increase backwashing efficiency by increasing the backwashing flux, DSobw with CaCl2 were used. However, this was inefficient due to interactions between Ca2+ in the DSobw and the adhered cells, even for just 1 min: for a 55.8 L h−1m−2 flux, 39% of removal was obtained for a 3 M CaCl2 DSobw when compared to 93% removal for 3 M NaCl for a 36 L h−1m−2 flux. Therefore, both adhesion and backwashing conditions are important for cleaning of FO membranes.