Fungi assisted microalgae bioflocculation is an emerging, efficient and cost-effective microalgal harvesting method, but no study has systematically evaluated and compared fungal spore-assisted (FSA) and fungal pellet-assisted (FPA) microalgal harvesting methods. In this study, harvesting Chlorella sp. cells by co-culture with Penicillium sp. spores or pellets was compared. Temperature, glucose concentration, pH and fungi:algae ratio were the critical parameters for harvesting efficiency. The highest flocculation efficiency (99%) of FSA method was achieved in 28 h at 40 °C, 160 rpm, 5 g glucose/L and 1.1 × 104 cells/mL (spore). FPA method can harvest 98.26% algae cells in 2.5 h at 34 °C, 160 rpm, pH 4.0 with the fungi:algae ratio of 1:2. The carbon input for FPA is only half of that for FSA. FPA takes less time and needs less glucose input compared with FSA and may be more promising to be further developed as an effective microalgae bioflocculation method.