A better understanding of the non-targeted (bystander) effects of radiation may have important implications with regards to radiation risk assessment, radiation protection, and targeted cancer therapy. In the present study, the direct and bystander effects of α-particle irradiation in immortalized human fibroblasts (F11hTERT) and breast cancer cells (MCF-7) was investigated. To ensure a more accurate dose delivery to these different cell lines, an existing 238Pu α-particle irradiator was improved by the addition of a collimator and the development of an analytical equation for calculation of the radiation dose to cells. The mean dose rate and α-particle fluence were calculated for each cell line by taking into consideration the size of their nuclei. Bystander effect experiments were performed by transferring medium from irradiated to unirradiated cells and by measuring micronucleus formation in the cells. Both the immortalized human fibroblasts and the breast cancer cells displayed a bystander effect. In conclusion, the broad-beam α-particle irradiator improved in this study represents a useful tool in the investigation of direct and non-targeted effects of α-particle radiation.