Ultrafiltration membrane harvesting of Scenedesmus acuminatus was tested using alternative feed (AF) directions, i.e., bottom feed-top feed cycle and traditional bottom feed (BF). Both operations were investigated to compare the membrane performance and membrane fouling in microalgal harvesting process by scanning electron microscope (SEM), confocal laser scanning microscopy (CLSM) and Fourier transform infrared (FTIR). The results showed that when the AF was used with and without backwashing, average flux increased by 27.9% and 17.9%, respectively, comparing with BF (68 L m−2 h−1) and the final dry weight reached 197 g L−1 and 175.8 g L−1, respectively. Microalgal cell deposition on AF membrane was reduced from 1.44 × 105 cell cm−2 on BF membrane to 7.12 × 104 cell cm−2 on AF membrane, according to SEM observation. The protein and polysaccharides on the AF membrane surface were also reduced according to CLSM and FTIR analysis. Fouling analysis along the fiber length revealed that fouling was most severe at the top section for BF as a result of a lower shear rate at the outlet. AF operation generated dynamic filtration by frequently switching flow directions, increasing the shear rate at both the top and bottom of the fibers, and therefore filtration and clean process simultaneously provided good performance. Display omitted •The hypothesis of membrane fouling differs along the fiber length was confirmed.•Dynamic filtration with alternative feed direction alleviated fouling.•Microalgal cell deposition and protein/polysaccharide attachment were reduced.•Scenedesmus acuminatus was concentrated 179 times (197 g L−1), flux of 87.0 Lm−2 h−1 was obtained.