Required field validator does not disable any other form controls when it fails the validation. Try this below sample
<asp:FileUpload ID="FileUpload1" runat="server" />
<asp:RequiredFieldValidator ID="rfvFileupload" ValidationGroup="validate" runat="server" Display="Dynamic"
ErrorMessage="* required" ControlToValidate="FileUpload1"></asp:RequiredFieldValidator>
<asp:TextBox ID="txtSubj" runat="server" ></asp:TextBox>
<asp:RequiredFieldValidator ID="rfvSubject" ControlToValidate="txtSubj" runat="server" Display="Dynamic"
EnableClientScript="true" ErrorMessage="* required" ValidationGroup="validate" />
<asp:Button ID="btnupload" runat="server" Text="Send" ValidationGroup="validate" onclick="btnupload_Click" />
<asp:Button ID="btncancel" runat="server" Text="Cancel" onclick="btncancel_Click" />