||One of the economical approaches to harvest mature microalgal biomass is through the attached growth system, in which the microalgal cells form attachment onto support material to facilitate its separation from the cultivation medium. Hence, the spent coffee grounds (SCG) was employed as the support material, proffering alimentation, and platform for Chlorella vulgaris sp. microalgae, to populate its surface. The SCG dosage of 8 g L−1 was revealed as optimum in enhancing the attached microalgal growth, i.e., achieving the microalgae density of 2.43 ± 0.04 g g-SCG−1 under continuous illumination. The introduction of a 20:4 photoperiod regime with the dark period cycle of 4 h day−1 and the remaining 20 h day−1 of illumination yielded a maximum microalgal density of 3.970 ± 0.8 g g-SCG−1. In this regard, the dark period had allowed the attached microalgae to strengthen their mixotrophic growth, i.e., assimilating more carbon from SCG effectively amidst heterotrophic mode. Besides, the extension of the dark period to 20 h day−1 had been identified as the threshold requirement to stress the attached microalgae in accumulating a significant amount of lipid which was about fivefold more productive than the attached microalgae growing under the continuous illumination. The fatty acid methyl esters (FAME) profile derived from harvested attached microalgal biomass was loaded primarily with alkyl chains of C12 to C18. Also, the high saturation degree of FAME had indicated the stability of biodiesel produced from attached microalgae growing onto SCG feedstock.