Microalgae-based biodiesel has gained widespread interest as an alternative energy source. Low-cost microalgae harvesting technologies are important for economically feasible biodiesel production. This study investigated, for the first time, the impact of adaptation period and height to diameter (H/D) ratio of a reactor on the growth and self-flocculation of microalgae, without the addition of bacteria. Six reactors were grouped into three sets of experiments, and each reactor was operated for 30 days at similar operating conditions (volume exchange ratio = 25% and settling time = 30 min). In set 1, two 8-L reactors, H5a (H/D ratio: 5) and H8a (H/D ratio: 8), were operated under batch operation. In set 2, reactors H5b and H8b were operated as sequential batch reactors (SBRs) without an adaptation period. In set 3, the reactors H5c and H8c were operated as SBRs with an adaptation period. The findings showed a threefold improvement in biomass productivity for the higher H/D ratio (H8c) and a reduction in biomass loss for microalgae. The H8c reactor exhibited 95% settling efficiency within 5 days, in comparison to 30 days for the H5c reactor. This study demonstrated that a higher H/D ratio and the introduction of an adaptation period in SBR operation positively influences growth and self-flocculation of enriched mixed microalgae culture.