The understanding that tumor cells might evade immunity through various mutations and the potential of an augmented immune system to eliminate abnormal cells led to the idea of utilizing platelet-rich fibrin (PRF), a blood concentrate containing the body's immune elements as an adjunctive therapy for localized tumors. This study is the first that evaluated the effect of PRF generated with different relative centrifugal forces (RCFs) on osteoblastic and fibroblastic tumor cell lines MG63 and HT1080 with regard to cell viability, cytokine and growth factor release, and the gene expression of factors related to the cell cycle and apoptosis. Our findings could demonstrate decreased cell proliferation of MG63 and HT1080 when treated indirectly with PRF compared to cell cultures without PRF. This effect was more distinct when the cells were treated with low-RCF PRF, where higher concentrations of growth factors and cytokines with reduced RCFs can be found. Similar patterns were observed when assessing the regulation of gene expression related to the cell cycle and apoptosis in both MG63 and HT1080 cells treated with PRF. Despite variations, there was a consistent trend of an up-regulation of tumor-suppressive genes and a down-regulation of anti-apoptotic genes in both cell types following treatment with high- and, particularly, low-RCF PRF formulations.