An ethnobotanical survey was carried out to collect information on medicinal plants and gender roles in sustainable use by urban communities of Morogoro and Iringa districts, Tanzania. A total of 99 and 75 medicinal plant species distributed in various genera and vascular plant families, curing about 72 and 57 human diseases, were reported in the two districts respectively. Results showed significant gender roles difference in sustainable use of medicinal plants. Most men herbalists used roots while women preferred barks. Uprooting during plant harvesting was agreed to exist by most men herbalists in Morogoro. The majority of men and women herbalists supported the idea of conserving medicinal plants in order to ensure their sustainability in future. Since medicinal plants play an important role in the livelihoods of urban dwellers, their loss due to unsustainable exploitation will obstruct the existing health care system unless conservation measures are taken. There is a need to sensitize and train herbalists and other users on sustainable ways of harvesting medicinal plants, encourage traditional conservation methods and domestication possibilities in home gardens. Emphasis on domestication should be a priority to women considering their marginal economic status and special interest in plant resources. Government legislation regarding plants protection should be strengthened to control harvesting of medicinal plants in the wild. Studies to validate safety and efficacy, assess harvesting sustainability and wild status of medicinal plants is recommended to improve the health care system and strengthen conservation abilities in Tanzania.