Inflammation mediated by the innate immune system is an organism’s protective mechanism against infectious environmental risk factors. It is also a driver of the pathogeneses of various human diseases, including cancer development and progression. Microalgae are increasingly being focused on as sources of bioactive molecules with therapeutic potential against various diseases. Furthermore, the antioxidant, anti-inflammatory, and anticancer potentials of microalgae and their secondary metabolites have been widely reported. However, the underlying mechanisms remain to be elucidated. Therefore, in this study, we investigated the molecular mechanisms underlying the anti-inflammatory and anticancer activities of the ethanol extract of the Antarctic freshwater microalga Micractinium sp. (ETMI) by several in vitro assays using RAW 264.7 macrophages and HCT116 human colon cancer cells. ETMI exerted its anti-inflammatory activity by modulating the main inflammatory indicators such as cyclooxygenase (COX)-2, interleukin (IL)-6, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and nitric oxide (NO) in a dose-dependent manner. In addition, ETMI exerted cytotoxic activity against HCT116 cells in a dose-dependent manner, leading to significantly reduced cancer cell proliferation. Further, it induced cell cycle arrest in the G1 phase through the regulation of hallmark genes of the G1/S phase transition, including CDKN1A, and cyclin-dependent kinase 4 and 6 (CDK4 and CDK6, respectively). At the transcriptional level, the expression of CDKN1A gradually increased in response to ETMI treatment while that of CDK4 and CDK6 decreased. Taken together, our findings suggest that the anti-inflammatory and anticancer activities of the Antarctic freshwater microalga, Micractinium sp., and ETMI may provide a new clue for understanding the molecular link between inflammation and cancer and that ETMI may be a potential anticancer agent for targeted therapy of colorectal cancer. The main purpose of this study was to assess the anti-inflammatory and cytotoxic effects of the ethanol extract from the polar microalga Micractinium sp. (ETMI) on the human colon cancer cell line, HCT116.