Apoptosis, also known as programmed cell death type I (PCD I), is a phenomenon responsible for the proper development and functionality of animal organisms. It enables the elimination of old, damaged, malfunctioning, oncogenic, and/or infected cells. As an essential determinant of organismal health, apoptosis is subject to multi-level regulation. PCD I can be induced via two molecular pathways: the extrinsic and the intrinsic. The latter is mitochondria-dependent – the key event in this signaling cascade is the permeabilization of the outer mitochondrial membrane (OMM), leading to the release of cytochrome c, apoptosome formation, the proteolysis of caspase-9, and, finally, the activation of caspase-3, an effector enzyme of apoptosis. Bid is a member of the Bcl-2 family, which comprises proteins responsible for regulating mitochondrial integrity. Its active form, truncated Bid (tBid), can promote OMM permeabilization, partly by recruiting Bax protein to the OMM, resulting in Bax oligomerization into pores that perforate the membrane. As Bid is cleaved to tBid downstream of surface receptor activation, it serves as a link integrating the extrinsic and intrinsic apoptotic pathways. Our study aimed to determine the influence of the ectromelia virus Moscow strain (ECTV-MOS) infection of L929 fibroblast on Bid activation and the consequent events, including colocalization of Bax with the OMM, caspase-3 activation, and changes in the structure and integrity of the cell membrane, measured cytometrically using an annexin–propidium iodide kit. We used Western blot to determine the level of Bid and active caspase-3, confocal microscopy to examine the Bax-OMM colocalization, and flow cytometry to assess the percentage of apoptotic cells in control as well as in ECTV-infected and/or staurosporine (STS)-treated fibroblasts. ECTV-MOS infection did not activate Bid, but counteracted STS-induced apoptosis by decreasing intracellular Bid levels, Bax-OMM colocalization, and caspase-3 activation. The results suggest that ECTV-MOS actively inhibits mitochondria-dependent PCD I.