Efficient cell disruption is critical for the recovery of lipids from microalgae for biodiesel production. The rupture of Chlorella sp. and Navicula sp. by high pressure homogenization (HPH) after acidic (HCl, to pH 1.5) and thermal (up to 123 °C) pretreatment was investigated quantitatively (cell counting) and qualitatively using scanning electron microscopy (SEM). Cell counting suggested that cell rupture by HPH was inhibited by each pretreatment, while SEM images showed that acidic and thermal pre-treatments damaged cell walls but caused the solidification of the cellular protoplasts which remained intact with HPH. The solidified protoplasts appeared similar to whole cells during cell counting and therefore did not register as ruptured; this limitation should be considered when using cell counting to determine the extent of cell rupture. Further investigations into thermally pretreated Chlorella sp. indicated that a monophasic lipid extraction was not hindered by protoplast solidification. The larger post-rupture cell fragments are hypothesized to offer potential benefits for downstream processing.