This study was adjusted to assess the cleaning efficacy of using 5% apple vinegar irrigation in adjunct with diode laser 980 nm in removing the smear layer and its effect on the atomic concentration of the calcium/phosphorus (Ca/P) ions by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectroscopy, respectively. A total of 60 maxillary incisor teeth were selected to be endodontically treated, prepared biomechanically, and divided equally into three main groups according to irrigation used—the control group—irrigation with 5% sodium hypochlorite (NaOCl) only, group I: irrigation with 5% NaOCl + 17% ethylenediaminetetraacetic acid (EDTA), group II: irrigation with 5% NaOCl + 5% apple vinegar. Diode laser 980 nm, 2 W/CW applied for 10 seconds. Three times in half the number of samples. All samples were divided longitudinally for SEM and SEM-EDX analysis at the coronal, middle, and apical root sections. Results showed group II had a statistically significant lower median smear layer score at the apical third (p < 0.05) when compared to group I and the control group. This study concluded that during root canal treatment, irrigation of 5% apple vinegar irrigation with 5% NaOCl in adjunct to diode laser 980 nm is better than17% EDTA and 5% NaOCl or 5% NaOCl alone in cleaning the intraradicular dentinal walls from the smear layer and also did not alter the atomic concentration of Ca/P ions ratio.