The prevalence of Helicobacter pylori resistance to levofloxacin and metronidazole was high in the Dominican Republic. We used two-fold agar dilution method to determine the minimum inhibitory concentration of five alternative antibiotics in 63 Dominican strains. We also assessed the genetic mutations associated with the antibiotic resistance using next-generation sequencing. We revealed that all 63 strains were sensitive towards sitafloxacin, furazolidone, and rifabutin. In contrast, the prevalence of rifaximin and garenoxacin resistance were high (82.5% and 34.9%, respectively). Patients more than or equal to 60 years old had the highest risk of double-antibiotic resistance (7/9, 77.8%, OR = 31.5, P = 0.009) and garenoxacin resistances (8/9, 88.9%, OR = 45.33, P = 0.002) with an increasing risk simultaneously by age (P = 0.004, r = 0.357). Almost all rifaximin resistant strains possessed multiple mutations with more than three mutations within rpoB including the most frequent novel mutations of S352L, I2726L, and V2465A. There was a significant association between vacA genotype and rifaximin resistance (P = 0.042). Among 23 levofloxacin-resistant strains, 82.6% (19/23, P <0.001) were also resistant to garenoxacin, and 39.1% (9/23) had a high minimal inhibitory concentration ≥8 μg/mL with positive trend correlation (P = <0.001, r = 0.84). Among 19 garenoxacin resistant strains, 16 (84.2%) contained mutations at D91 and N87 of gyrA. In conclusion, sitafloxacin, rifabutin, and furazolidone might be considered as alternative antibiotics to be included in H. pylori eradication regimen in regions with high prevalence of levofloxacin and metronidazole resistance, such as the Dominican Republic.