Background: The dose-related effects of patient setup errors on bio-physical indices were evaluated for the conventional wedge (CW) and field-in-field (FIF) whole breast irradiation techniques (WBI). Materials and Methods: The treatment plans of 10 patients receiving left WBI were retrospectively selected for evaluation. The bio-physical effects of dose variations were evaluated by shifting the isocenters and gantry-angles of the treatment plans. Dose-volume histograms of the planning target volume (PTV), heart, and lungs were generated, and the conformity index (CI), homogeneity index (HI), tumor control probability (TCP), and normal tissue complication probability (NTCP) were determined. Results: The D95 of the PTV for an “isocenter shift plan” with a posterior direction decreased by approximately 15%, and the TCP of the PTV decreased by approximately 50% for the FIF technique and by 40% for the CW; however, the NTCPs of the lungs and heart increased for both techniques. Increasing the gantry-angle decreased the TCPs of the PTV by 24.4% (CW) and by 34% (FIF). The NTCPs of the lungs and heart for the two techniques differed by only 3%. The CIs and HIs for the CW case were higher than the corresponding values obtained for the FIF case. Significant differences were observed between the two techniques (p<0.01). Conclusion: Our results revealed that the biophysical properties of the FIF case were more sensitive to setup errors than those in the CW case. The radiobiological-based analysis could be detected significant dosimetric errors and provided a practical patient quality assurance method for guiding the bio-physical effects.